Abstract HIV-1 persists in memory CD4+ T cells during suppressive antiretroviral therapy (ART) and is the major barrier to virus cure. Ultimately, a successful HIV eradication approach will need to work equally well regardless of disease stage, underlying immune system function, or medical comorbidities. People with HIV are commonly exposed to opioids, whether prescribed for chronic pain, prescribed for opioid use disorder, or injected as heroin. Opioid exposure has demonstrated immune modulatory effects but the impact of opioids on HIV-1 reservoir dynamics has not been studied and thus there is an urgent need for focused, mechanistic studies to address this critical knowledge gap. The purpose of this proposal is to apply cutting-edge experimental approaches to understand precisely how opioids and opioid use disorders impact HIV-1 reservoir dynamics and latency reversal and transform our ability to study and test HIV-1 eradication approaches in this important patient population. The scientific premise of this proposal is that opioids limit HIV-1 transcriptional and translational reactivation due to a previously unrecognized block in RNA and protein synthesis. While the HIV eradication field has focused on transcriptional biomarkers of latency reversal, our preliminary ribosome profiling data reveals that translation, not transcription, is the rate-limiting step in HIV-1 reactivation. Ribosome profiling is a novel technique that uses deep sequencing to characterize the nuclease- protected footprints of ribosomes on mRNA transcripts in vivo. Protein synthesis can be monitored genome-wide and when combined with mRNA sequencing (mRNA-seq) provides a quantitative measure of translation efficiency. We propose to leverage HIV and host genomic information and investigate the mechanisms that control HIV-1 latency reversal in patients using opioids. We hypothesize that opioid use will limit HIV-1 latency reversal and lower HIV-1 translation efficiencies, when compared to HIV-1 latency reversal in the absence of opioid exposure. To define the effects of opioids on traditional HIV-1 persistence markers in vivo, we will perform a cross-sectional analysis among HIV-infected treated suppressed participants and quantify and compare traditional HIV-1 persistence markers from 5 groups: individuals who are 1) actively injecting opioids (n=20), 2) on methadone maintenance (n=20), 3) on buprenorphine maintenance (n=20), 4) on prescribed opioids for chronic pain treatment (n=20), and 5) on no opioids (n=40). We will perform ex vivo reactivation experiments with participants' PBMC and a panel of LRA. LRA-induced levels of HIV-1 caRNA and supernatant virion production will be compared across opioid use groups. To accurately measure genome-wide host and HIV-1 translation in PBMC isolated from patients who use opioids, parallel mRNA-seq and ribosome profiling will be performed. Finally, we will assess longitudinal HIV-1 reservoir dynamics in a group of 40 participants during the transition from active injection opioid use to buprenorphine for opioid use disorder.